3 | DISCUSSION
For ultrasound-induced endosomal escape, both destabilization of the endosomal membrane and protein release from the carriers are required. In this study, to meet the former requirement, PCNDs were employed as an ultrasonically endosome-disruptive carrier based on a pioneering study[28]. To meet the later requirement, a disulfide-linked material for the bio-reductive release of proteins was developed in this study. This material was confirmed to conjugate with a model protein with the lipid coating of droplets and to release it in response to reductive conditions in vitro (Figure 2A). Even in living cells, the bio-reductive release of β-Gal and Sap was strongly suggested by the hydrolysis of the fluorogenic substrate in the cytosol (Figure 3) and enhancement in cytotoxicity (Figure 4), respectively. Furthermore, cytosolic delivery of these two model proteins was observed in an ultrasound-responsive manner (Figure 3 and 4). This result indicates that endosomal escape of cargo proteins was achieved by vaporization of PCNDs.
In our previous works, intracellular vaporization of antibody-conjugated PCNDs was utilized for ultrasound-dependent induction of cell death to targeted cells[30,31]. Different from the present study, the PCNDs which included the equal mixture of PFH (boiling point = 57 °C) and perfluoropentane (PFP, boiling point = 29 °C) was employed. Here, employment of inner perfluorocarbons with a low boiling point increases the size and the lifetime of bubbles after vaporization, leading to remaining and coalescence of bubbles[28]. Such properties of PCNDs with a lower boiling point are desirable in intracellular vaporization for enhancing cytotoxicity, and therefore, the PCNDs including the PFH-PFP mixture was employed in the previous works[30,31]. In the present study for cytosolic protein delivery, the PCNDs including PFH only was employed for suppressing cytotoxicity of their vaporization. Actually, in β-Gal delivery, no significant cell damage was observed in the microscopic images one hour after ultrasound exposure (Fig. 3), as observed in the pioneering study in which the PCNDs including PFH only were used[28]. This result is quite different from the images in our previous works in which cells were disrupted and detached from the dish surface by PCND vaporization[30]. However, in Sap delivery, the vaporization of PCNDs without carrying Sap caused non-negligible cytotoxicity after incubation for 48 h (Figure 4). In our previous work, the cell viability after vaporization of PCNDs was influenced by the PCND concentration during cellular uptake and the experimental setup for ultrasound exposure[31]. Further optimization of these conditions is required to manipulate cell functions without causing cell damage. Thus, although there is a room for improvement, we have demonstrated a proof-of-principle that ultrasound-dependent cytosolic protein delivery is possible using chemically functionalized PCNDs.
In conclusion, the protein-conjugated PCNDs could be used for ultrasound-induced cytosolic protein delivery. Through the development of a thiol-reactive lipid coating, cargo proteins were carried on the droplets through a bio-reductive disulfide linkage. When the cargo protein had no thiol group, it was conjugated with the droplets through reversible thiolation of the amine groups. In principle, the proposed method can be applied to any protein. These protein-conjugated PCNDs were taken up into living cells, and after ultrasound exposure, the cargo proteins functioned in the cytosol in an ultrasound-induced manner, probably because of endosomal escape by the vaporization of the droplets in the endosomal pathway. In this study, by utilizing an anti-cancer antibody as the molecular device for intracellular delivery, protein-conjugated PCNDs were accumulated in cancer cells. Similarly, according to the objectives, proteins can be selectively delivered into the cytosol of various targeted cells by employing the desired targeting ligands. Furthermore, based on the double targeting effect of both ultrasound and the ligands, the current method of cytosolic delivery is expected to meet a wide variety of demands in fundamental studies and therapeutic uses.